RNA interference (RNAi) is a natural and robust gene silencing process in animal cells that investigators can harness to silence the expression of practically any gene of interest. The power and versatility of RNAi stems from the relative ease of synthesizing and introducing into cells small RNAs with an enormous range of sequences that efficiently incorporate into the RNA Induced Silencing Complex (RISC) as Guide strands. Guide strands direct the RISC to specific complementary transcripts, and RISC then triggers catalytic inhibition of translation or degradation of the complementary transcripts. The RNAi methodology has revolutionized many areas of biology because it has enabled highly specific reverse genetics analyses in cultured animal cells, and become an essential tool in studying diseases from cancer to diabetes to neurological disorders.
In vertebrate cells, the biological function of the RNAi pathway is to regulate the expression of the transcriptome through microRNAs (miRNAs). Genome-encoded miRNA genes are transcribed by RNA-Polymerase (Pol) II as a long primary transcript (pri-miRNA) which is processed into a small hairpin-shaped precursor (pre-miRNA) by the Drosha enzyme, and then further processed by the Dicer enzyme into a small duplexed RNA. The resulting small RNA duplex is then sampled on both ends of the duplex for ease in unwinding the RNA ends according to the thermodynamics of RNA base-pairing. Thus, a single-stranded guide RNA, such as a mature miRNA sequence, becomes incorporated into an Argonaute protein that is the core effector protein of the RISC. The RISC can then bind transcripts, with as few as 7-8 nt of complementarity with the ‘seed’ sequence in the 5′ end of the guide RNA, to trigger inhibition of translation and promote mRNA destabilization. However, complete complementarity through the entire length of the guide RNA can trigger catalytic cleavage of the mRNA and lead to more robust gene silencing.
Investigators harness RNAi by programming the RISC with two types of triggers: chemically synthesized small interfering RNA (siRNA) or vector-driven transgenes that express a short hairpin RNA (shRNA). Although still extensively used, siRNAs have the restrictions of higher cost and shorter-lived gene knockdown effects because the siRNA is diluted by cell division and RNA turnover. shRNAs were originally used by transfecting cells with a plasmid that expressed a short transcript driven by RNA Pol III type of promoters to make a small fold back structure which would be short enough not to trigger undesired innate immunity response.
Currently, due to unpredictability with respect to silencing efficacies and potential off-targeting effects from each individual shRNA, researchers targeting individual genes with shRNAs must obtain a panel of multiple shRNA constructs. In some instances an entire panel of shRNAs may fail. Failure is likely due to competition with the endogenous miRNA pathway, unpredictable guide versus passenger strand production, secondary structures and/or RNA binding proteins that occlude the RNAi machinery from accessing the mRNA. Thus, there is a continuing need for improved shRNAs with increased efficacy and reliability.